This invention is in the field of ex vivo culture of hematopoietic cells, more specifically in the culture of human dendritic cells and T-cells for therapeutic purposes.
Dendritic cells play an important role in immune responses. They provide the immune system with an effective means of antigen presentation, unlike that of any other cell. Dendritic cells are the most potent antigen presenting cells in the immune system. They have been characterized to have a unique morphology and cell surface phenotype that may contribute to their potency in initiating cellular immune responses, specifically T cell dependent responses. Therefore, dendritic cells have been proposed as a valuable component in cellular based therapies that require presentation of antigen to effector cells. Dendritic effector cells can be primed ex vivo to create activated cells to be reintroduced into the body in order to combat disease. However, dendritic cells comprise less than 1% of leukocytes circulating in peripheral blood, which makes it difficult to obtain an amount sufficient to use in therapy.
Dendritic Cells (DC) along with monocytes/macrophages and B lymphocytes are considered professional antigen-presenting cells (APC); (reviews by Caux et al., Immunology Today 16: 2-4, 1995; Steinman, Annu. Rev. Immunol9: 271-296, 1991; Young, J W, et al., Stem Cells 14:376-387, 1996; Steinman, R M, Exptl Hematol 24:859-882, 1996). Even though antigen-presenting cells take in, process, and present antigen to T lymphocytes, they serve different immune functions. DC are the most potent initiators of the immune response and are the APCs responsible for the induction of primary antigen-specific immune reactions (Bhardwaj et al., J. Exp. Med. 175: 267-273, 1992; Bhardwaj et al., J. Exp. Med. 178: 633-642, 1993). The DC, as are all hematopoietic cells, are derived from CD34+ stem cells. These hematopoietic cells arise in the bone marrow and as they mature traffic to the peripheral blood where they circulate (T cells make a detour to the thymus) and then may enter tissues. The differentiation pathway from a CD34+ cell to a DC is not completely understood (Santiago-Schwarz et al., J. Leukoc. Biol. 52: 274-281, 1992; Galy et al., Immunity 3: 459-473, 1995; Rosenzwajg et al., Blood 87: 535-544, 1996). DC are found in very low levels in peripheral blood ( less than 1%) compared to other white blood cells (WBC) or leukocytes (neutrophils about 60%, lymphocytes about 35%, monocytes about 5%, eosinophils about 2%, basophils  less than 1%); (Laboratory Medicine Hematology, editor: J. Miale, publisher: C.V. Mosby Co., 1982). Thus, in order to use DC therapeutically, it would be most practical to expand them ex vivo. To date, the culture of DC cells has been performed in serum-containing media and semi-closed tissue culture plates or flasks. To expand DC under these conditions, GM-CSF appears to be important for DC differentiation, and addition of TNF-xcex1 appears to be inhibitory to other CD34+ progenitors as greater DC are seen in those cultures (Santiago-Schwarz et al., Blood 82: 3019-3028, 1993). Addition of IL-4 causes the down regulation of CD14+ monocytes (Sallusto et al., J. Exp. Med. 179: 1109-1118, 1994; Romani et al., J. Exp. Med. 180: 83-93, 1994). Thus cultures that included those cytokines have shown preferential outgrowth of DC.
To date, DC have been identified by a bundle of criteria that cover different features of DCxe2x80x94morphology, phenotype, and their function in mixed lymphocyte reactions (MLR) have been evaluated (O""Doherty et al., J. Exp. Med., 178: 1067-1078, 1993; Freudenthal and Steinman, PNAS 87: 7698-7702, 1990; Crow et al., Clin. Exp. Immunol. 49: 338-346, 1982; Caux et al., J. Exp. Med. 180: 1841-1847, 1994).
Morphologically, DC are identified by their cytoplasmic processes or xe2x80x9cveilsxe2x80x9d that extend from the surface of the cells. The unusual morphology of DC has been described in many publications and illustrated in photomicrographs. For example, Young and Steinman describe DC thusly: xe2x80x9cDC extend long processes, easily 10xcexc in length, in many directions from the cell body. In the living state, these processes are sheet-like xe2x80x9cveilsxe2x80x9d or lamellipodia and are actively motile. When spun onto glass slides, the DC processes are numerous and spinyxe2x80x9d. (Young, J W, et al., i Stem Cells 14:376-387, 1996.)
Phenotypically, DC constitutively express CD80 and CD86, the costimulatory molecules that bind CD28 on T cells. The ability to stimulate T-cells in an allogeneic mixed lymphocyte reaction is a functional capacity attributed to DC. It has recently been shown that DC are very efficient presenters of antigen and can result in potent CD8+ cytotoxic T cell responses (Bender et al., J. Exp. Med. 182: 1663-1671, 1995; Tjoa et al., The Prostate 28: 65-69, 1996).
It has been shown that DC can be culture derived from renal cell carcinoma patients (Radmayr et al., Int. J. Cancer 63: 627-632, 1995). When DC are isolated from prostate cancer patients that have undergone various immunocompromising therapies, the DC are functional after being in culture ex vivo (Tjoa et al., The Prostate 27: 63-69, 1995).
Ronald Levy""s group has reported remissions in B-cell lymphoma patients treated with autologous DC which had been isolated from their peripheral blood (Hsu, F. J., et al, Nature Medicine 2:52-57, 1996). The monocyte-depleted leukapheresis product was initially cultured for 24 hours with an idiotype protein surface antigen specific to each patient""s tumor; then DC were purified by differential gradient centrifugation, and returned to culture for an additional 14-18 hours prior to intravenous administration of the DC.
DC culture to date has been performed in medium containing cytokines such as granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-4 (IL-4), tumor necrosis factor-xcex1 (TNF-xcex1), and stem cell factor (SCF). The development of recombinant forms of these cytokines in recent years has made their use in the clinical setting much more practical than it would otherwise have been when they were only available in small quantities purified from natural sources. However, recombinant proteins are expensive, and not always available in a form needed for clinical use, i.e. produced under Good Manufacturing Conditions (GMC). Moreover, clinical trials are required to obtain regulatory approval for the clinical use of each individual cytokine.
There have been reports in the literature of using small molecules in attempts to promote differentiation of hematopoietic cells other than DC. T and B cells were stimulated with L-leucine methyl ester, which caused the death of monocytes (Thiele, et al. 1983 J Immunol. 131:2282-2290).
Thiele et al. also reported using mitogens such as plant lectins, concanavalin A, pokeweed, sodium periodate, and neuraminidase plus galactose oxidase. Terminal monocyte differentiation in cell lines was potentiated by the combination of transforming growth factor-xcex2 plus Vitamin D3 (Testa, et al. J Immunol. 1993 150:2418-2430). Vitamin D2 was reported to increase differentiation of monocytes in a cell line, while retinoic acid did not (Howell, et al. 1994 Blood Coagulation and Fibrinolysis 5:445-453). Leukemia cell lines were reported to increase their differentiation to mature granulocytes, or monocytes/macrophages, or erythroid cells upon stimulation with tubulin disruptors, TPA, retinoids, or hemin (Nakajima, et al. 1994 Biol. Pharm. Bull. 17:742-744). The HL60 cell line was reported to show increased differentiation towards neutrophils or monocytes upon stimulation with retinoids and Vitamin D3 (Bunce et al. 1995 Leukemia 9:410-418).
It would be most advantageous to be able to culture DC without the use of cytokines, or with only one cytokine, using only common molecules which are readily available, inexpensive, and possibly already approved for use in the clinical setting.